Chemical Ionization Spectra of Permethyl Glycosylalditols (3) R. F. Schaub and M. J. Weiss, J. Am. Chem. Soc., 80, 4683 (1958). (4) J. A. Wright, N. F. Taylor, and J. J. Fox, J. Org. Chem., 34, 2632 (1969). (5) S. R. Jenkinsand E. Walton, Carbohydr. Res., 26, 71 (1973).
(6)A. Yamashita and A. Rosowsky,unpublished results. (7) A. Rosowsky in "HeterocyclicCompoundswith Three- and Four-Membered Rings", A. Weissberger, Ed., Interscience, New York, N.Y., 1964, pp 394-418.
(8) A.-M.
Sepulchre,G. Lukacs, G. Vass, and S.D. Gero, Bull. Soc. Chim. Fr.,
4000 (1972).
(9) A.-M. Sepulchre,A. Gateau-Olesker,G. Vass, and S.D. Gero, Biochlmie, 55, 613 (1973). (IO) H. Paulsen, V. Sinnwell,and P. Stadler, Chem. Ber., 105, 1978 (1972). (1 1) E. J. Corey and D. Seebach,Angew, Chem., lnt. Ed. Engl., 4, 1075 (1965);
J . Org. Chem., Vol. 41, No. 21,1976
3425
D. Seebach, ibid., 8, 639 (1969). (12) C. D. Anderson, L. Goodman,and B. R . Baker, J. Am. Chem. Soc., 80, 5247 (1958). (13) H. Kuzuhara and S. Emoto, Agric. Biol. Chem., 28, 900 (1964). (14) J. A. Montgomeryand S.D. Clayton,J. Carbohydr.,Nucleosides, Nucleotides, 2, 147 (1975). (15) E. J. Reist and S. L. Holton, Carbohydr. Res., 9, 71 (1969). (16) T. Van Es, Carbohydr.Res., 21, 156 (1972). (17) J. A. Montgomery, M. C. Thorpe, S.D. Clayton, and H. J. Thomas, Carbohydr. Res., 32, 404 (1974). (18) H. N. Schleln,M. Israel, S.Chatterjee,and E. J. Modest, Chem. lnd. (London), 418 (1964). (19) J. A. Wright and N. F. Taylor, Carbohydr. Res., 6, 347 (1968).
Polysaccharide Sequencing by Mass Spectrometry: Chemical Ionization Spectra of Permethyl Glycosylalditolsl 0. S.Chizhov,* V. I. Kadentsev, and A. A. Solov'yov Zelinsky Institute for Organic Chemistry, Academy of Science, Moscow, U.S.S.R.
P. F. Levonowich and R. C. Dougherty* Department of Chemistry, Florida State University, Tallahassee, Florida 32303 Received February 18,1976 Chemical ionization (CI) mass spectra with isobutane and isobutane/ammonia as the reagent gases are reported for the six permethylated glucosylalditols and for two permethylated biosylalditols. Intense peaks corresponding to MH+ or (M + NH4)+ were observed in all cases. In the isobutane CI spectra the ratio of abundances for the aldito1 ions that were formed by cleavage of the glycosidic bond on the alditol or glucosyl side of the glycosidic oxygen 144,and 1-3 the depended strongly on the position of the glycosidic linkage. When the linkage was 1-6,l-2, ratio of intensities for the altitol+ (A+) and alditol hydrate+ (AOH2+) ions were respectively 0.72, 0.37, 0.17, and 0.06. The fragmentation of a biosylalditol (7) was clearly consistent with the relative intensities for the A+ and AOH2+ ions anticipated from the disaccharide results. The ratio of A+ to AOH2+ was 0.16 for this 1-4 linked aldito1 Determination of the structure of oligosaccharides by mass spectrometry is directly analogous to the problem of sequencing polypeptides by the same technique. However, in the case of carbohydrates the details of the structure are considerably more subtle. Ideally one would like to obtain information about the molecular weight, subunit structure, and position sequence. Fortunately conventional techniques will give reliable information concerning subunit structure in unknown oligosaccharides. The information that must be obtained from the mass spectrum is thus reduced to molecular weight and structure and position sequence. Chemical ionization (CI) mass spectra of oligosaccharide peracetates using ammonia/isobutane2 and isobutane3 alone as reagent gases have been investigated in this regard. The primary reagent ion in isobutane CI mass spectra is the tert-butyl cation (C4Hgf).4Proton transfer from this ion is considerably more exothermic than proton transfer from the ammonium ion or attachment of NH4+ to a molecule. The ammonium ion is the dominant reagent ion in ammonia/isobutane CI mass ~ p e c t r a .The ~ , ~higher exothermicity of ionization in isobutane CI mass spectra results in extensive fragmentation of oligosaccharide peracetates so that the spectra resembled the electron ionization (EI) mass spectra of these molecules in many details.3 In particular the intensity of ions with masses near the molecular weight of the molecule were quite low in both E1 and isobutane CI mass spectra. It has been possible to obtain sequence information from E1 mass spectra of permethylated sugars.6 The low intensity of the high mass ions is a distinct disadvantage in these cases and the high energies associated with the ionization process tend to cloud the structural information in the spectra.
Ammonia/isobutane chemical ionization mass spectra of oligosaccharide peracetates gave molecular weight and structure sequence information for di-, tri-, and tetrasaccharides.2 The dominant fragment ions in these spectra corresponded to ammonium ion attachment to thermolysis fragments. The thermolysis fragments reliably produced information concerning the masses of the subunits and their sequence. However, it has not yet been possible to determine positions for the subunits in the chain from these spectra. The permethyl derivatives of oligosaccharides are considerably less polar and more volatile than their acetate analogues. Thus it seemed reasonable to expect2 that these derivatives might be used to give position sequence information in CI mass spectra. We have used the permethylated alditol derivatives because reduction of the carbonyl terminus of the oligosaccharide prior to methylation unequivocally tags the reducing end of the sugar for sequence analysis. In this report we discuss the isobutane and ammonia/isobutane CI mass spectra of six permethylated glycosylalditols and two permethylated biosylalditols. The numerical designations, names, and molecular weights for these compounds are indicated in Table I.
Results and Discussion Isobutane CI Spectra. The major ions in the isobutane CI mass spectra of the glucosylalditols, 1-6, are presented in Table 11. The most intense ion in every case corresponded to the protonated molecule. All six of the compounds in Table I1 showed very similar fragmentation patterns. Probable structures for the major ions and routes for their formation are illustrated for permethy~-2-0-~-D-g~ucopyanosyl-D-g~~-
3426 J . Org. Chem., Vol. 41, No. 21,1976
Chizhov, Kadentsev, Solov'yov, Levonowich, and Dougherty
Table I. Names and Molecular Weights of Permethylated Glyosylalditols Examined in This Study Compd
Name
Mol wt
470 471 471 471 470 1-0-a-L-arabinopyranosyl-DL-xylitol 382 a-D-G~ucopyranosy~-l+4-~-a-D-glucopyran-674 osyl-1~4-0-D-glucito1 a-D-Glucopyranosyl-l-+4-0 -@-D-glucopyran- 630 osyl-1-1-0-DL-xylitol 2-O-@-D-G~ucopyranosyl-D-g~ucito~ 2-O-@-D-G~ucopyranosyl-D-g~ucitol-l -d 3-0 -~-D-G~ucopyranosyl-D-g~ucito~-l -d 4-0-a-D-Glucopyranosyl-D-glucitol-l -d 6-O-@-D-Glucopyranosyl-D-glucitol
CH,OMe
oMe
'I
b
CHzOMe
A', m/e 235
MH', m/e 471
citol(1) in Scheme I. In the spectrum of 1 and the rest of the glucosylalditols there are two major series of fragment ions, one derived from the glucosyl portion of the molecule-G+, (G - CH30H)+, [G - (CH30H)z]+-and the other derived from the alditol portion of the molecule-AOHz+, A+, (A CH30H)+. Since the glucopyranose end group was the same in all cases one would not expect the ions in the glucosyl series to carry significant information concerning the linkage position to the alditol. This is not true of the alditol ion series and indeed the relative intensities of alditol, A+, and alditol hydrate ions, AOH2+, do vary strongly with the linkage position to the alditol. In the series 5,2,4, and 3 in which the glycosidic linkage is 1-6, 1-2, 1-4, and 1-3 the ion intensity ratio for A+/ AOH*+ was respectively 0.72,0.37,0.17, and 0.06. Repeated measurements as well as the comparison between the spectra of 1 and 2 indicate that the reproducibility of these ratios is roughly &15%.Since the ratios differ by roughly a factor of 2 in all cases it should be possible to reliably use the ratio of the A+ (mle 235) to AOH2+ (mle 253) ions as an indication of the terminal linkage position in reduced and permethylated polysaccharides. The results for the 1 4 4 linked biosylalditol, 7, are in complete agreement with this expectation (ratio of A+ to AOH2+ 0.16). The intensity ratio for the A+ (mle 191) and AOH2+ (mle 209) for the 1-1 linked arabinosyl xylitol, 6, was 0.65. This value is very to the ratio of A+/AOH2+ for the terminally linked glycosyl alditol, 5 (0.72). The intensity ratio for the same ions for the xylitol containing trisaccharide, 8, was 0.67, which further supports the utility of these ions in assignments of position sequence in premethylated alditols. The formation of the A+ ions in these spectra is easily un-
Hot J
G+, m/e 219
CH20Me
AOH2+,m/e 253
[G-MeOH]', m/e 1187
m/e 439 OMe MHI+,m/e 471 derstood by postulating protonation on the glycosidic oxygen followed by assisted cleavage to give the alditol cation. The ease of protonation as well as cleavage should be dependent on the linkage position in the alditol. Protonation on the glycosidic oxygen must also be responsible for the presence of the glucosyl ions G+ and their fragments in the spectra. The AOH2+ ions must arise by protonation at a site remote from the glycosidic oxygen7p8followed by elimination of the
Table 11. Monoisotopic Isobutane CI Mass Spectra of Permethylated Glycosylalditolsa MH MH+ MeOH)+ AOH2+
Compd 1
mle
471
re1 intensity
100
439 9 440 20 440 10 440 6 439 10 351 3
(G (A (G MeOH)+ MeOH)+ 2MeOH)+
A+
G+
253
235
219
187
155
25
10
1
7
10
Other ions 509 3 502 2.5 509 1.5 509 1.5 509 1.0
194 2 263
89 2 89
155 80 mle 472 254 236 219 187 2 4 4 15 13 2 13 re1 intensity 100 38 155 296 113 101 3 mle 472 254 236 219 187 1.5 1.5 3 12 40 2.5 2.5 re1 intensity 100 16 4 mle 472 254 236 219 187 155 296 93 4 1 8 2.5 2 1.5 re1 intensity 100 24 5 mle 471 253 235 219 187 155 295 re1 intensity 100 3.5 2.5 0.5 3.5 3 3 6 mle 383 209 191 175 143 159 111 277 245 211 201 re1 intensity 100 13 20 9 2 1 1.5 2.5 4 1.5 5 mle 193 177 145 2 1.5 5 re1 intensity "Adduct ions, e.g., (M C4Hg)+,whose intensity strongly depends on the precise conditions in the source have not been reported. The same is true for ions with less than 1%relative abundance. bA = alditol moiety, G = glycosyl moiety. 2
+
Chemical Ionization Spectra of Permethyl Glycosylalditols
J . Org. Chem., Vol. 41, No. 21,1976 3427
m/e Figure 1. Isobutane CI mass spectrum of c u - D - g ~ u c o p y r a n o s y ~ - ~ ~ ~ - ~ - a - D - g ~ u c o p y r a n o s y ~ - ~(7). ~~-~-D-g~ucito~
40
#
155
.
100
150
200
300
anosyl-1- 1 0 -DL-xylitol(8).
Figure 2. Isobutane CI mass spectrum of a-D-glucopyr
~
glucosyl residue with transfer of a hydrogen atom to the alditol fragment. The metastable ion that appeared in all of the spectra of 1-5 at mle 136.2 unequivocally connects the AOH2+ ion with the protonated molecule. The fact that we were unable to detect a metastable ion for the reaction AOH2+ A+ H20 supports the contention that the precursor to the AOH2+ ion is not protonated on the glycosidic oxygen and thus elimination of water from AOH2+ would require a proton rearrangement. The isobutane CI mass spectra of the biosylalditols 7 and 8 are illustrated in Figures 1 and 2. In both cases the protonated molecule is a prominent ion in the spectrum. The two ion types seen in the glucosylalditol spectra, namely G+ and A+, are the prominent fragment ion series in both of these spectra. The fragments in the G series (mle 155,187,219,and 423) and the A series (mle 235, and 457) for 7 and mle 191,209, and 413 for 8 permit unequivocal determination of the mass sequence of the residues in the chain. The origins of the G and A series ions in these spectra are indicated in Scheme 11. Other than the sequence ions, the spectra of 7 and 8 both contain a prominent ion whose origin is not obvious. The prominent ion a t mle 471 in the spectrum of 7 corresponds to a protonated permethyl glucosylalditol. The ammonia/iso-
-
+
Scheme I1
OMe
G+, m/e 219
GIG2, m/e 423 CH@Me 7
G,'
m/e 219
G'G~,m/e 423 8
OMe CH,OMe
butane mass spectrum as well as GLC analysis gave no evidence of contamination of 7 with a permethylated disaccharide and thus this ion must have arisen by methoxy or methyl transfer in cleavage of the terminal glucosyl residue. The spectrum of 8 contains a prominent ion at mle 279 which does
/
CHBO
not belong to the sequence ion series. The mass and isotope ratio suggested a formula for this ion of C12H2307 which would be compatible with a bicyclic structure involving the central glycosyl unit. Again the ammonialisobutane spectra and GLC both indicated that the rnle 279 ion was not an artifact. Ammonia/Isobutane CI Mass Spectra. The ammonia1 isobutane mass spectra of this series of compounds have fewer prominent ions than their isobutane analogues in agreement with our expectations. The prominent ions in the ammonia1 isobutane mass spectra of 1-8 are recorded in Table 111. These spectra are generally dominated by even mass ammonium ion complexes of molecules. Thus the fragments must have arisen by pyrolytic destruction of the original sugar derivatives. The fragments in the spectra of the biosylalditols exemplify the reactions that occur. The ion at mle 472 in the mass spectrum of 7 would correspond to the ammonium ion complex of a permethyl glucosylglucopyranose.This molecule was probably formed by methyl or methoxyl transfer in the cleavage of the alditol terminus. The lower intensity of the same ion in the spectrum of 8 is consistent with structure dependence of the cleavage rate. The fact that the ion appears in both spectra indicates that it does not contain the alditol portion of the molecule. The ion at mle 428 in the spectrum ~ , , : which could arise by a of 7 corresponds to C I ~ H ~ ~ ONH4+ complex thermal fragmentation process removing C1 from the central glucosyl unit along with its attached alditol. The mle 400 ion contains one less CO than that a t 428, and could be a product of a similar fragmentation. Proton transfer fragment ions also appear in the spectra as indicated in Table 111;however, the intensity of these ions as
3428 J . Org. Chem., Vol. 41, No. 21,1976
Chizhov, Kadentsev, Solov'yov, Levonowich, and Dougherty
Table 111. Monoisotopic Ammonia/Isobutane CI Mass Spectra of Permethylated Reduced Di- and Trisaccharides" Compd
(M+NH4)+
MH+
(AOH+2NH4)+
AOH2+
A+
Other ions
mle re1 intensity mle re1 intensity
488 270 253 235 450 439 100 4 2.5 8 5.5 3 2 489 472 271 236 280 155 100 1 2 2.5 2.5 1 3 mle 489 472 271 254 236 296 210 re1 intensity % 100 4.5 9.5 1.5 5.5 4 5 4 mle 489 472 254 236 110 re1 intensity 100 3 1 2 2 5 mle 488 471 270 253 235 110 156 re1 intensity 100 1.5 4.5 1 1 15 8 6 mle 400 383 191 236 re1 intensity 100 2 1.5 5 7 mle 692 488 486 472 428 400 re1 intensity 100 12 14 25 20 8 8 mle 648 414 408 302 296 157 re1 intensity 100 16 4.5 3.5 11 5 aOnly the six most intense ions have been reported. The spectra were recorded with relatively low concentrations of the substrate in the source so that (2M NH4)+ ions were insignificant. 1
+
well as that of the thermal fragment ions was low and generally of less structural utility than the corresponding ions in the isobutane spectra. Summary. Isobutane CI mass spectra of permethylated saccharide alditols offer considerable promise for obtaining detailed oligosaccharide sequence information. Molecular weights and the mass sequence of monosaccharide units may be obtained directly from the spectra. The linkage position t o a glucitol terminus may be inferred by the intensity ratios of alditol and alditol hydrate ions. On this basis the prospects for obtaining other position sequence information by examination of these spectra seems good.
Experimental Section The permethylated oligosaccharide alditols used in this study were prepared by standard method^.^ The deuterium labeled derivatives were prepared by NaBD4 reduction of the parent oligosaccharide. Mass spectra were obtained by use of a solid probe inlet with an AEI MS-902 mass spectrometer equipped with an SRIC chemical ionization source. Spectra were recorded at a source pressure of 0.5 Torr (160 Pa). The source temperature was 150 "C. Ammonia/isobutane spectra were obtained with a 2:l mixture of ammonia and isobutane at a total pressure of 0.5 Torr. When isobutane was used as the reagent gas the intensity of the tert- butyl cation (mle 57) exceeded the intensity of the most intense
ion in the rest of the spectrum by at least a factor of 10. The recorded spectra were all obtained with the I3C ion to rnle 58 off scale on the least sensitive range. In the ammonia/isobutanespectra the ammonium ion (mle 18) was always at least 10 times the intensity of any of the other ions in the spectrum. Registry No.-1, 30608-25-4; 2, 30608-28-7; 3, 59907-27-6; 4, 30608-29-8; 5, 29923-20-4; 6, 59907-28-7;7, 32581-17-2; 8, 5990729-8. References a n d Notes (1) This work was supported in part by a grant from the National Science Foundation. (2) (a) 0.S. Chizhov, V. I. Kadentsev, A. A. Solov'yov, W. W. Binkley, J. D. Roberts, and R. C. Dougherty, Dokl. Akad. Nauk SSSR, 217,362 (1974); (b) R. C. Dougherty, J. D. Roberts, W. W. Binkley, V. I. Kadentsev, A. A. Solov'yov, and 0. S. Chizhov, J. Org. Chem., 39, 451 (1974). (3) D. Horton, J. D. Wander, and R. Foltz, Carbohydr. Res., 38, 75 (1974). (4) H. M. Fales, G. W. A. Milne, and R. S. Nicholson, Anal. Chem., 43, 1785 (1971). (5) A. M. Hogg and T. L. Nagabhushan, Tetrahedron Lett., 4827 (1972). (6) (a) N. K. Kochetkov, N. S. Wulfson, 0. S. Chizhov, and B. M. Zolotarev, Tetrahedron, 19, 2209 (1963); (b) N. K. Kochetkov and 0. S. Chizhov, ibid., 21, 2029 (1965); (c) M. J. M. Colavd, P. A. Dumont, and F. Compernolle, Biomed. Mass Spec., 2, 156 (1975). (7) M. S. B. Munson, Anal. Chem., 43, 28A (1971). (8) G. W. A. Milne, T. Axenrod. and H. M. Fales, J. Am. Chem. SOC.,92,5170 (1970). (9) R. W. Bailey, "Oligosaccharides", Pergamon Press, Elmsford, N.Y., 1965.
